The protozoan parasite Plasmodium falciparum is responsible for the deadliest form of human malaria, with complications including coma, anemia, respiratory distress, and renal failure. With no effective vaccine at hand, chemotherapy remains the cornerstone for malaria treatment and control. Efforts to eradicate malaria have been hindered by the rise of resistance to chloroquine (CQ), an otherwise safe, cheap, and highly effective drug. CQ resistance (CQR) is primarily mediated by the P. falciparum Chloroquine Resistance Transporter (PfCRT). In addition to mediating CQR, PfCRT is a modulator of parasite susceptibility to first-line artemisinin- based combination therapies (ACTs), whose efficacy is endangered by emerging multidrug resistance (MDR) in Southeast Asia. While all geographically-distinct CQR parasites have acquired multiple single-nucleotide polymorphisms (SNPs) in PfCRT, presumably to balance their requirements for drug resistance and fitness, we are currently unable to explain temporal changes in the prevalence of different regional PfCRT haplotypes. To understand how specific PfCRT SNPs affect parasite drug resistance and fitness (Aims 1 and 2), we will genetically dissect two CQR pfcrt alleles, Ecu1110 (from Ecuador) and Cam734 (from Cambodia). Ecu1110 pfcrt contains 4 SNPs, the fewest observed, rendering it amenable to a study of the mutational trajectories that lead to acquisition of drug resistance. Cam734 pfcrt, the most highly mutated allele yet described, harbors 5 novel SNPs and serves as a prototype for the study of the unique gain of parasite fitness afforded by pfcrt. We will also explore the capacity of PfCRT SNPs to facilitate resistance to first-line ACT components (Aim 3), namely artemisinins (ARTs) and their partner drugs, an effort that will combine our laboratory-based investigations with on-site clinical training in western Cambodia, an epicenter of MDR. In summary, our aims are (1) to investigate the contribution of PfCRT SNPs to the development of resistance to CQ and other clinically significant antimalarials, (2) to evaluate the impact of PfCRT SNPs on parasite fitness, and (3) to assess the capacity of PfCRT to modulate susceptibility to ARTs and ACT partner drugs in multidrug-resistant parasites. These studies are expected to yield important new insights into the genetic basis of antimalarial drug resistance, including the role of pfcrt mutations in modulating the efficacy of first-line combination therapie, and will guide the development of novel strategies to reduce the global impact of multidrug-resistant malaria.